We are continuing to investigate the mechanisms by which adeno-associated satellite viruses (ASV) interfere with the replication and potential oncogenesis of their helper adenoviruses and herpes viruses. Production of type 4 ASV structural antigens and infectious DNA was studied using temperature-sensitive (ts) mutants of herpes simplex virus (HSV) types 1 and 2. Mutants belonging to three HSV-1 and two HSV-2 complementation groups complemented ASV antigen production, while members of two other HSV-1 groups did not. Production of HSV DNA, HSV-induced DNA pllymerase and thymidine kinase do not appear necessary viral functions to establish ASV antigen complementation. However, a correlation exists between the ability of herpes mutants to synthesize HSV structural proteins and their ability to complement ASV antigen production. Infectious S16 ASV DNA was isolated from cells coinfected with a temperature-sensitive mutant of herpes simplex virus (HSV) type 1 in the absence of contaminating HSV DNA. This satellite virus DNA does not differ in its physical, chemical and biological properties from DNA isolated directly from virions or from cells coinfected with adenovirus. Single-stranded ASV DNA from alkaline sucrose gradients has a modal length of 1.5 micromicron and demonstrates evidence of internal redundancies in the electron microscope.